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Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives
Accepted Manuscript Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives Zhong-Hua Li, Tao-Qian Zhao, Xue-Qi Liu, Bing Zhao, Chao Wang, Peng-Fei Geng, Ya-Quan Cao, Dong-Jun Fu, Li-Ping Jiang, Bin Yu, Hong-Min Liu PII:
S0223-5234(17)30837-1
DOI:
10.1016/j.ejmech.2017.10.037
Reference:
EJMECH 9829
To appear in:
European Journal of Medicinal Chemistry
Received Date: 22 June 2017 Revised Date:
11 October 2017
Accepted Date: 13 October 2017
Please cite this article as: Z.-H. Li, T.-Q. Zhao, X.-Q. Liu, B. Zhao, C. Wang, P.-F. Geng, Y.-Q. Cao, D.-J. Fu, L.-P. Jiang, B. Yu, H.-M. Liu, Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.10.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphic abstract
Pteridin-7(8H)-ones were synthesized and evaluated for their antiproliferative activity.
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Compound 12 displayed acceptable growth inhibition, induced apoptosis of MKN-45 cells. Besides, a novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold was first prepared.
ACCEPTED MANUSCRIPT Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives Zhong-Hua Li a, b, c, d, 1,Tao-Qian Zhao a, b, c, d, 1, Xue-Qi Liu a, b, c, d, Bing Zhao a, b, c, d, Chao Wang a, b, c, d, Peng-Fei Geng a, b, c, d, Ya-Quan Cao a, b, c, d, Dong-Jun Fu a, b, c, d,
a
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Li-Ping Jiang e, Bin Yu a, b, c, d,*, Hong-Min Liu a, b, c, d,* Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan
Province;
b
Key Laboratory of Technology of Drug Preparation (Zhengzhou University),
Ministry of Education of China; c Key Laboratory of Henan Province for Drug Quality and
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Evaluation; d School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China; e School of Basic Medical Science，Central South University, Changsha 410013,
*Corresponding Authors: Prof. Hong-Min Liu & Dr. Bin Yu
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PR China
Zhengzhou University, Zhengzhou, 450001, China
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E-mail: [email protected] (Hong-Min Liu) & [email protected] (Bin Yu)
ABSTRACT: Pteridines are an important class of heterocyclic compounds with diverse biological activities. Here, we report a series of pteridin-7(8H)-one derivatives
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and their antiproliferative activities toward MKN-45, MGC-803, EC-109, and H1650. Structure-activity relationship studies showed that compound 12 exerted the most
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potent antiproliferative activity against MKN-45 and MGC-803 with the IC50 values of 4.32 and 7.01 µM, respectively. Besides, compound 12 induced morphological changes and apoptosis of MKN-45 cells, increased expression of Bax, down-regulated expression of Bcl-2 and caused cleavage of caspase-3/9. Additionally, we first reported the construction of the novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and a chiral carbon center established. Keywords: Pteridin-7(8H)-one, Antiproliferative activity, Apoptosis, Cell cycle arrest
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ACCEPTED MANUSCRIPT 1. Introduction Pteridines are an important class of bicyclic N-heteroarenes, which have received considerable attention from medicinal community due to their diverse biological activities, such as antimicrobial [1], antiallergic [2], immunosuppressive [3],
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anti-inflammatory [4, 5], antibacterial [6, 7], and anticancer activities [8, 9]. Some biologically active natural products (e.g. folic acid, etc.) are also found to possess pteridine scaffolds. As shown in Fig. 1, folic acid (also named as vitamin B9 or vitamin M) is necessary for human health and is involved in DNA synthesis, DNA
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repair and DNA methylation [10]. Antifolate methotrexate (MTX) is a chemotherapy agent and immune system suppressant for treatment of cancer and autoimmune
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diseases . Moreover, pteridine analogs are well known to act upon a wide range of targets for therapeutic potential [4]. Compound A, as a selective PI3K/mTOR dual inhibitor, is highly efficacious in mouse xenograft model bearing glioma cell line U87 [11]. Compound B has been proved to be able to inhibit EGFRwt and EGFRT790M/L858R potently and exerts remarkable in vivo growth inhibition of human non-small cell lung
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cancers (NSCLC) [12]. Compound C is a pteridinone-based TLR7 (Toll-like receptor 7) agonist, which is currently in clinical evaluation for the treatment of chronic HBV (hepatitis B) infection [13] (Fig. 1). The diverse bioactivities and natural prevalence
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make pteridines and their structural analogs promising bicyclic scaffolds to develop new agents for the treatment of different diseases.
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Following our previous work on the synthesis of new bicyclic heterocycles with anticancer potentials [14-17], we herein report the synthesis of a series of new pteridin-7(8H)-one derivatives through cascade reactions, their antiproliferative activity and preliminary mechanisms of inducing cancer cell death. Also, we first report the synthesis of new 8,9-dihydro-7H-purine-8-carboxylate via the 5-endo cyclization.
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2. Results and discussion 2.1 Chemistry
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Fig. 1. Biologically active pteridine derivatives.
The general synthetic route is illustrated in Scheme 1. The intermediate derivatives 2a-h were synthesized by condensation of commercially available pyrimidine analogs
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1a-c with appropriate primary amines in the presence of N,N-diisopropylethylamine (DIPEA) in DMF. The obtained intermediates 2a-h reacted with ethyl glyoxalate in AcOH/EtOH under heating to afford pteridin-7(8H)-one skeletons 3a-h [11], which
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then reacted with various amines in the presence of TEA in a mixed solvent of ethanol
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and DMF, generating the target compounds 4-21.
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ACCEPTED MANUSCRIPT O Cl
Cl NH2
N R1
R2CH2NH2
1a-c
NH2
N
DMF, DIPEA, 100 oC, 4 h
Cl
N
N
R1
H
AcOH/ethanol (1 : 1) 100 oC, 2h
N
N
O R2
N H
Cl OEt
N
N
R1
3a-h
2a-h
1a, R1 = propyl-S1b, R1 = Me1c, R1 = H-
O R2
2a-3a, R1 = propyl-S-, R2 =Ph2b-3b, R1 = propyl-S-, R2 =PhC2H4-
N
1
R
Ethanol/DMF (3 : 1) reflux, 3h
2e-3e, R1 = propyl-S-, R2 =
N
N
R'RNH/TEA
2d-3d, R1 = H-, R2 =Ph-
R'
2f-3f, R1 = propyl-S-, R2 = N 4-21
N
S
O
2g-3g, R1 = propyl-S-, R2 =
R2
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R
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2c-3c, R1 = Me-, R2 =Ph-
O
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2h-3h, R1 = propyl-S-, R2 =
Scheme 1. Synthesis of pteridin-7(8H)-one derivatives.
Interestingly, compound 3h' was also observed in the conversion from compound 2h to
compound
3h.
Compound
3h'
features
a
novel
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8,9-dihydro-7H-purine-8-carboxylate scaffold. Both compounds were formed through the unstable imine intermediate A. For the synthesis of compound 3h, it involved the 6-exo cyclization (Scheme 2, path b), generating one C-N bond, one C=N bond, and the pteridin-7(8H)-onescaffold. While compound 3h' was competitively formed via
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the 5-endo cyclization (Scheme 2, path a), accompanying by the formation of two C-N bonds, a chiral center, and the 8,9-dihydro-7H-purine-8-carboxylate architecture.
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To the best of our knowledge, this is the first report on the rapid construction of the new
8,9-dihydro-7H-purine-8-carboxylate
framework,
which
also
possesses
pluripotent groups (e.g. –COOEt, Cl) for further structural derivations. Besides, the C-N single bond of compound 3h' could be potentially oxidized to aromatic bicyclic N-heteroarene.
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Scheme 2. Proposed reaction mechanism for the synthesis of compounds 3h and 3h'.
Compound 3h' was then characterized by 2D NMR spectra (see supporting
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information for details). Intriguingly, the methylene protons (highlighted in red) appeared at 3.99 and 3.17 ppm, respectively. The unique intramolecular H-bond interactions and the adjacent chiral center may be responsible for the remarkably different chemical shifts of the methylene protons.
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2.2 Evaluation of biological activity 2.2.1 Antiproliferative activity
Compounds 4-21 were evaluated for their antiproliferative activity against MKN-45
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and MGC803 (human gastric cancer cell lines), EC-109 (human esophageal cancer cell line), and H1650 (human lung cancer cell line), by using the MTT assay, and
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5-fluorouracil (5-FU) was employed as the reference drug [18]. The preliminary data are summarized in Tables 1-3.
As shown in Table 1, compounds 4-6 with different aniline substituents exerted moderate antiproliferative activity against the tested cancer cell lines, the substituents attached had certain effect on the activity, especially for MKN-45 and MGC-803 cells. Compound 6 with the 3,4,5-trimethoxyl group was more potent than compound 4 with the IC50 values of 14.25 and 15.03 µM, respectively against MKN-45 and MGC-803 cells. Compounds 7, 8, 13 and 14 with the aliphatic amine substituents showed 5
ACCEPTED MANUSCRIPT decreased inhibitory activity against the tested cancer cell lines. Compound 12 was significantly more potent than compounds 9-10, highlighting the importance of nitrogen atom of cyclic amine groups for the activity. Compound 11 with a benzyl group attached to the piperazine nitrogen atom showed decreased activity compared
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to compound 12. Compound 12 displayed good and broad-spectrum antiproliferative activities with IC50 values less than 10 µM, comparable to that of 5-FU.
Table 1
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Inhibitory activity of compounds 4-14 against the tested cancer cell lines
IC50 (µM)a
Compound
R'RN-
MGC-803
EC-109
H1650
38.60±3.75
36.60±4.69
19.03±1.38
>64
14.65±1.80
19.35±0.56
>64
51.84±3.86
14.25±1.42
15.03±1.86
38.67±1.56
36.94±0.96
7
>64
46.64±2.56
>64
>64
8
21.02±1.16
30.40±2.54
36.04±0.98
>64
9
>64
49.64±0.86
>64
>64
10
>64
34.71±1.43
>64
54.80±2.43
11
15.43±2.47
18.71±5.69
>64
>64
5
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6
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4
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MKN-45
6
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4.32±1.87
7.01±3.61
9.85±2.81
9.92±1.41
13
39.23±4.72
>64
>64
>64
14
23.26±1.86
43.74±1.80
29.71±1.65
>64
8.89±1.65
7.52±0.98
6.21±0.75
14.25±2.73
a
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-
5-Fu
Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs
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of three independent experiments.
Next, the influence of R2 substituents on the activity was also investigated. As shown
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in Table 2, compounds 15-19 generally displayed decreased or comparable activities toward the tested cancer cells, regardless of the patterns of R2 group.
Table 2
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Inhibitory activity of compounds 15-19 against the tested cancer cell lines
MKN-45
MGC-803
EC-109
H1650
15
9.97±0.97
13.75±3.81
18.28±2.95
>64
16
11.32±1.57
16.41±3.16
19.69±6.07
>64
17
11.56±6.50
>64
24.04±4.88
54.91±6.08
Compound
R
IC50 (µM)a
2
7
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17.92±2.82
53.67±5.66
>64
51.81±3.21
19
16.83±5.65
17.17±2.35
37.48±4.62
45.43±2.90
8.89±1.65
7.52±0.98
6.21±0.75
14.25±2.73
5-Fu
Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
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a
-
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent experiments.
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However, the replacement of propylthio group with methyl (compound 20) or hydrogen (compound 21) led to the loss of antiproliferative activity against the tested
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cancer cell lines, significantly less potent than 5-FU and compound 12 (Table 3).
Table 3
R
a
IC50 (µM)a
1
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Compound
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Inhibitory activity of compounds 20-21 against the tested cancer cell lines
MKN-45
MGC-803
EC-109
H1650
12
propyl-S-
4.32±1.87
7.01±3.61
9.85±2.81
24.91±4.41
20
Me-
>64
>64
38.98±0.99
>64
21
H
>64
>64
41.66±5.02
>64
5-Fu
-
8.89±1.65
7.52±0.98
6.21±0.75
14.25±2.73
Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent experiments. 8
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2.2.2 Cell apoptosis of MKN-45 and possible mechanism involved The acceptable antiproliferative activity of compound 12 promoted us to investigate its effect on the apoptosis and morphological changes. Hochest 33258 staining was
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performed to investigate morphological changes of MKN-45 cells [19]. After 24 h incubation with compound 12 at indicated concentrations, characteristic apoptotic morphological changes were observed, including cell rounding, chromatin shrinkage and formation of apoptotic bodies (Fig. 2A). Notably, this phenomena were more
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remarkable at higher concentrations. To further explore the effect of compound 12 on cell apoptosis, the apoptotic analysis was also performed with Annexin V-FITC/PI
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double staining and analyzed with flow-cytometry calculation [20]. Treatment of MKN-45 cells with compound 12 resulted in a concentration-dependent apoptosis
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increase (Fig. 2B/2C).
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Fig. 2. Compound 12 induced apoptosis of MKN-45 cells. (A) Apoptosis analysis with Hoechst
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33258 staining; (B) Apoptosis effect on MKN-45 cell line induced by compound 12 for 24 h using Annexin V-FITC/PI double staining and flow-cytometry calculation. The lower left quadrant
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represents live cells, the lower right is for early/primary apoptotic cells, upper right is for late/secondary apoptotic cells, and the upper left represents cells damaged during the procedure; (C) Quantitative analysis of apoptotic cells. The experiments were performed three times, and a representative experiment is shown.
Next, the western blot analysis was performed to examine the expression of apoptosis-related proteins. As shown in Fig. 3A, treatment of MKN-45 cells with compound 12 increased expression of Bax in a concentration-dependent manner (Fig. 3B). Bax was able to activate the caspases, and promoted the release of cytochrome c 10
ACCEPTED MANUSCRIPT and other pro-apoptotic factors from the mitochondria [21]. Meanwhile, the expression of anti-apoptotic protein Bcl-2 decreased accordingly (Fig. 3C). As shown in Fig. 3D and 3E, treatment of compound 12 also concentration-dependently caused the cleavage of caspases-3 and caspases-9, which then inducing cell apoptosis. These
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results indicated that compound 12 may induce MKN-45 apoptosis through the
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intrinsic apoptotic pathway.
Fig. 3. Expression changes of apoptosis-related proteins induced by compound 12. (A) Compound 12 induced expression changes of Bax, Bcl-2 and caspase family members in MKN-45 cells; (B-E) Statistical analysis of expression levels of Bax, Bcl-2 and cleaved-caspased 9/3.
2.2.3 Cell cycle analysis The effect of compound 12 on the cell cycle was also evaluated. After treatment of MKN-45 cells with compound 12 for 24 h at indicated concentrations (0, 2, 4, 8 µM), 11
ACCEPTED MANUSCRIPT the percentage of cells in G2/M phase were 1.62%, 16.6%, 29.2% and 40.8%, respectively (Fig. 4), suggesting that compound 12 caused an obvious G2/M arrest in a concentration-dependent manner with concomitant decrease in terms of the number
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of cells in other phases of the cell cycle.
Fig. 4. Effect of compound 12 on the cell cycle distribution of MKN-45 cells. Cells were treated with indicated concentrations of compound 12 (0, 2, 4, 8 µM) for 24 h. Then the cells were fixed and stained with PI to analyze DNA content by flow cytometry. The experiments were performed three times and a representative experiment is shown.
3. Conclusions In summary, a new series of pteridin-7(8H)-one derivatives were prepared and evaluated for their antiproliferative activity. Among them, compound 12 exhibited the 12
ACCEPTED MANUSCRIPT most potent and broad-spectrum inhibition against the tested cancer cells (MKN-45, MGC803, EC-109 and H1650) and displayed comparable activity with the anticancer drug 5-FU. Besides, compound 12 induced morphological changes and apoptosis of MKN-45 cells in a concentration-dependent manner, increased expression of
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pro-apoptotic protein Bax, down-regulated expression of anti-apoptotic protein Bcl-2 and caused cleavage of caspase-3/9, indicating that compound 12 induced apoptosis via the intrinsic apoptotic pathway. The G2/M phase arrest induced by compound 12 was also observed. Additionally, we first reported the construction of the novel
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bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and a chiral carbon center established.
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This scaffold contains several pluripotent handles, which allows for further structural modifications for identifying potential bioactive molecules.
4. Experimental section 4.1 General
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Reagents and solvents were purchased from commercial sources and were used without further purification. Melting points were determined on an X-5 micromelting apparatus and are uncorrected. 1H NMR and
13
C NMR spectra were recorded on a
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Bruker (DPX-400) spectrometer, operating at 400 and 100 MHz, respectively. High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-Tof of
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Micromass spectrometer by electrospray ionizaton (ESI).
4.2 General procedure for the synthesis of compounds 3h', 4-21. A solution of 2a (1eq) with ethyl glyoxalate (50 wt. % in toluene, 1.2 eq) in a mixed solvent of AcOH/ethanol (1 : 1) was heated to 100 oC for 2 h. After the completion of the reaction monitored by TLC (PE/EA = 4 : 1), the purification was conducted by flash column chromatography on silica gel using PE/EA (4 : 1) as eluent to afford 3a. As a representative example, the new compound 3h' was separated and characterized by NMR. Compounds 4 can be readily obtained by refluxing 3a (1eq), aniline (1.2 eq) and triethylamine (1.2 eq) in the mixed solvent of ethanol/DMF (3 : 1) for 3 h, 13
ACCEPTED MANUSCRIPT following by purification on flash column chromatography on silica gel. This procedure was also applied to preparation of compounds 5-21.
4.2.1 Ethyl (R)-6-chloro-9-(cyclohexylmethyl)-2-(propylthio)-8,9-dihydro-7H-purine-
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8-carboxylate (3h') Pale yellow solid, yield 40%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 5.10 (s, 1H), 3.96-4.01 (m, 1H), 3.60-3.66 (m, 2H), 3.5-3.20 (m, 1H), 3.04-3.11 (m, 1H), 2.93-3.00 (m, 1H), 1.61-1.73 (m, 8H), 1.14-1.20 (m, 3H), 1.09 (t, J = 7.0 Hz, 3H),
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0.98 (t, J = 7.3 Hz, 3H), 0.90-0.96 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.3, 160.2, 150.0, 141.1, 112.2, 85.9, 64.4, 52.2, 36.1, 32.3, 30.1, 30.1, 26.0, 25.3, 25.3,
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22.6, 14.9, 13.3. HR-MS (ESI): Calcd. C18H27ClN4O2S, [M+H]+m/z: 399.1621, found: 399.1623.
4.2.2 8-Benzyl-4-(phenylamino)-2-(propylthio)pteridin-7(8H)-one (4) Pink solid, yield 75%. 1H NMR (400 MHz, Chloroform-d) δ 8.42 (s, 1H), 7.98 (s, 1H),
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7.75 (m, 1H), 7.49-7.51 (m, 2H), 7.36-7.40 (m, 2H), 7.23-7.34 (m, 3H), 7.12-7.16 (m, 1H), 5.51 (s, 2H), 3.13 (t, J = 7.2 Hz, 2H), 1.73-1.83 (m, 2H), 1.04 (t, J = 7.3 Hz, 3H). 13
C NMR (100 MHz, Chloroform-d) δ 173.50, 156.99, 155.61, 147.37, 144.76,
137.85, 135.89, 129.01, 128.51, 127.85, 124.11, 120.41, 110.37, 43.77, 33.43, 22.99,
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13.61.HR-MS (ESI): Calcd. C22H21N5OS, [M+K]+m/z: 442.1104, found: 442.1102.
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4.2.3 8-Benzyl-4-((2-methoxyphenyl)amino)-2-(propylthio)pteridin-7(8H)-one (5) Yellow solid, yield 81%. 1H NMR (400 MHz, Chloroform-d) δ 9.15 (s, 1H), 8.62-8.64 (m, 1H), 8.02 (s, 1H), 7.49 (m, 2H), 7.27-7.33 (m, 2H), 6.93-7.10 (m, 3H), 5.52 (s, 2H), 3.97 (s, 3H), 3.15 (t, J = 7.4 Hz, 2H), 1.75-1.83 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H).
13
C NMR (100 MHz, Chloroform-d) δ 173.36, 157.03, 155.39, 148.79,
147.26, 144.77, 135.96, 128.97, 128.49, 127.80, 127.67, 123.54, 120.78, 119.99, 110.99, 110.12, 55.90, 43.74, 33.43, 23.00, 13.63.HR-MS (ESI): Calcd. C23H23N5O2S, [M+Na]+m/z: 456.1470, found: 456.1472. 14
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8-Benzyl-2-(propylthio)-4-((3,4,5-trimethoxyphenyl)amino)pteridin-7(8H)-one
(6) Yellow solid, yield 86%. 1H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.99 (s, 1H), 7.47-7.49 (m, 2H), 7.28-7.32 (m, 3H), 7.07 (s, 2H), 5.53 (s, 2H), 3.90 (s, 6H),
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3.85 (s, 3H), 3.15 (t, J = 7.3 Hz, 2H), 1.72-1.77 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 173.46, 156.97, 155.48, 153.36, 147.30, 144.76, 135.84, 134.77, 133.88, 128.80, 128.52, 127.85, 110.35, 98.32, 61.02, 56.21, 43.83, 33.40, 22.56, 13.49. HR-MS (ESI): Calcd. C25H27N5O4S, [M+Na]+m/z: 516.1681,
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found: 516.1682.
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4.2.5 8-Benzyl-4-(benzylamino)-2-(propylthio)pteridin-7(8H)-one (7)
Yellow solid, yield 68%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.48 (m, 2H), 7.35 (m, 4H), 7.26-7.31 (m, 4H), 6.78 (t, J = 5.6 Hz, 1H), 5.49 (s, 2H), 4.77 (d, J = 5.9 Hz, 2H), 3.09 (t, J = 7.3 Hz, 2H), 1.69-1.78 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13
C NMR (100 MHz, Chloroform-d) δ 173.22, 158.01, 157.14, 147.19, 144.21,
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137.88, 136.05, 128.92, 128.78, 128.46, 127.76, 127.70, 110.33, 44.80, 43.75, 33.39, 22.88, 13.58. HR-MS (ESI): Calcd. C23H23N5OS, [M+Na]+m/z: 440.1521, found:
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440.1524.
4.2.6 8-Benzyl-4-((cyclohexylmethyl)amino)-2-(propylthio)pteridin-7(8H)-one (8) White solid, yield 76%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.46-7.49
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(m, 2H), 7.27-7.31 (m, 2H), 7.22-7.25 (m, 1H), 6.57 (t, J = 6.1 Hz, 1H), 5.48 (s, 2H), 3.42 (t, J = 6.5 Hz, 2H), 3.09 (t, J = 7.4 Hz, 2H), 1.62-1.80 (m, 8H), 1.16-1.30 (m, 3H), 0.96-1.05 (m, 5H).
13
C NMR (100 MHz, Chloroform-d) δ 173.05, 158.35,
157.20, 147.03, 143.84, 136.12, 128.94, 128.43, 127.71, 110.35, 47.04, 43.69, 37.99, 33.37, 30.88, 26.37, 25.83, 23.01, 13.62. HR-MS (ESI): Calcd. C23H29N5OS, [M+Na]+m/z: 446.1991, found: 446.1992.
4.2.7 8-Benzyl-4-morpholino-2-(propylthio)pteridin-7(8H)-one (9) 15
ACCEPTED MANUSCRIPT Yellow solid, yield 79%. 1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.45 (m, 2H), 7.29 (m, 2H), 7.23 (m, 1H), 5.53 (s, 2H), 4.27-4.30 (m, 4H), 3.80 (t, J = 4.7 Hz, 4H), 3.05 (t, J = 7.3 Hz, 2H), 1.68-1.77 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 171.09, 157.27, 156.17, 149.70, 141.83, 136.15, 128.73,
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128.43, 127.66, 112.36, 67.09, 47.94, 44.09, 33.29, 22.85, 13.56. HR-MS (ESI): Calcd. C20H24N5O2S, [M+Na]+m/z: 420.1470, found: 420.1471.
4.2.8 8-Benzyl-2-(propylthio)-4-thiomorpholinopteridin-7(8H)-one (10)
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White solid, yield 81%. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.46 (m, 2H), 7.24-7.31 (m, 3H), 5.52 (s, 2H), 4.49 (t, J = 4.9 Hz, 4H), 3.05 (t, J = 7.3 Hz, 2H), 13
C NMR (100
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2.76 (t, J = 5.0 Hz, 4H), 1.70-1.76 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).
MHz, Chloroform-d) δ 171.06, 157.23, 156.13, 149.80, 141.94, 136.17, 128.82, 128.42, 127.68, 112.34, 50.70, 44.09, 33.31, 27.62, 22.91, 13.58. HR-MS (ESI): Calcd. C20H23N5OS2, [M+Na]+m/z: 436.1242, found: 436.1245.
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4.2.9 8-Benzyl-4-(4-benzylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (11) White solid, yield 72%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.45 (m, 2H), 7.23-7.34 (m, 8H), 5.51 (s, 2H), 4.28 (m, 4H), 3.54 (s, 2H), 3.04 (t, J = 7.3 Hz,
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2H), 2.56 (t, J = 5.0 Hz, 4H), 1.67-1.76 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.88, 157.15, 156.22, 149.68, 141.48, 137.60, 136.25, 129.21, 128.79, 128.40, 128.33, 127.62, 127.27, 112.35, 62.96, 53.31, 47.44, 44.04,
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33.28, 22.89, 13.56. HR-MS (ESI): Calcd. C27H30N6OS, [M+H]+m/z: 487.2280, found:487.2281.
4.2.10 8-Benzyl-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (12) White solid, yield 82%. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.44-7.46 (m, 2H), 7.28-7.31 (m, 1H), 7.22-7.26 (m, 2H), 5.52 (s, 2H), 4.30 (m, 4H), 3.05 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 4.9 Hz, 4H), 2.34 (s, 3H), 1.68-1.77 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.64, 156.60, 155.33, 149.36, 141.68, 16
ACCEPTED MANUSCRIPT 136.17, 128.19, 127.33, 127.06, 111.54, 54.65, 46.89, 45.50, 43.49, 32.45, 22.48, 13.18. HR-MS (ESI): Calcd. C21H26N6OS, [M+H]+m/z: 411.1967, found: 411.1968.
4.2.11 8-Benzyl-4-(propylamino)-2-(propylthio)pteridin-7(8H)-one (13)
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Yellow solid, yield 85%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 6.0 Hz, 1H), 7.96 (s, 1H), 7.24-7.31 (m, 5H), 5.35 (s, 2H), 3.38-3.43 (m, 2H), 2.99 (t, J = 7.2 Hz, 2H), 1.54-1.66 (m, 2H), 0.86-0.92 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.85, 153.02, 151.93, 141.78, 138.66, 130.86, 123.64, 123.17, 122.44, 105.10, 38.44, 37.37,
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28.12, 17.71, 17.54, 8.32, 6.16. HR-MS (ESI): Calcd. C19H23N5OS, [M+Na]+m/z:
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392.1521, found:392.1522.
4.2.12 8-Benzyl-4-(cyclopropylamino)-2-(propylthio)pteridin-7(8H)-one (14) Yellow solid, yield 68%. 1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 4.5 Hz, 1H), 7.95 (s, 1H), 7.22-7.32 (m, 5H), 5.36 (s, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.96 (m, 1H), 1.58-1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H), 0.68-0.74 (m, 4H). 13C NMR (100 MHz,
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DMSO-d6) δ 171.37, 158.96, 156.27, 146.85, 143.68, 136.24, 128.30, 127.30, 127.13, 110.00, 43.14, 32.51, 24.04, 22.63, 13.24, 5.98. HR-MS (ESI): Calcd. C19H21N5OS,
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[M+Na]+m/z: 390.1365, found: 390.1366.
4.2.13 4-(4-Methylpiperazin-1-yl)-8-(3-phenylpropyl)-2-(propylthio)pteridin-7(8H)one (15)
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Yellow solid, yield 63%. 1H NMR (400 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.28 (m, 1H), 7.25 (m, 1H), 7.15-7.21 (m, 3H), 4.35-4.39 (m, 2H), 4.31-4.32 (m, 4H), 3.03 (t, J = 7.2 Hz, 2H), 2.73 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 5.1 Hz, 4H), 2.36 (s, 3H), 2.03-2.08 (m, 2H), 1.74-1.79 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.96, 157.26, 156.20, 149.64, 141.53, 141.08, 128.35, 128.23, 125.95, 112.34, 54.96, 46.94, 45.58, 41.08, 33.34, 33.32, 28.72, 22.92, 13.56. HR-MS (ESI): Calcd. C23H30N6OS, [M+H]+m/z: 439.2280, found: 439.2281.
4.2.14 4-(4-Methylpiperazin-1-yl)-2-(propylthio)-8-(thiophen-2-ylmethyl)pteridin17
ACCEPTED MANUSCRIPT 7(8H)-one (16) Yellow waxy solid, yield 78%. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.34-7.36 (m, 1H), 7.14 (d, J = 3.5 Hz, 1H), 6.92-6.94 (m, 1H), 5.53 (s, 2H), 4.21-4.23 (m, 4H), 3.12 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 5.0 Hz, 4H), 2.23 (s, 3H), 13
C NMR (100 MHz, Chloroform-d) δ
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1.70-1.77 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H).
170.97, 157.15, 155.63, 149.19, 141.53, 137.29, 128.87, 126.39, 126.10, 112.28, 55.14, 47.15, 45.81, 38.42, 33.34, 22.88, 13.62. HR-MS (ESI): Calcd. C19H24N6OS2,
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[M+H]+m/z: 417.1531, found: 417.1532.
4.2.15 8-(Furan-2-ylmethyl)-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-
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one (17)
Yellow waxy solid, yield 73%. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.55 (d, J = 1.7 Hz, 1H), 6.39 (m, 1H), 6.29 (d, J = 3.2 Hz, 1H), 5.38 (s, 2H), 4.18 (m, 4H), 3.04 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 5.0 Hz, 4H), 2.20 (s, 3H), 1.63-1.73 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, Chloroform-d) δ 171.04, 157.20, 155.80,
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149.37, 142.06, 141.56, 112.27, 110.41, 109.42, 55.06, 47.08, 45.72, 37.04, 33.33,
401.1762.
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22.94, 13.58. HR-MS (ESI): Calcd. C19H24N6O2S, [M+H]+m/z: 401.1760, found:
4.2.16 8-(Cyclohexylmethyl)-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)one (18)
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White solid, m. p. 146-147 oC, yield 72%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 4.29 (m, 4H), 4.19 (d, J = 7.3 Hz, 2H), 3.07 (t, J = 7.4 Hz, 2H), 2.53 (t, J = 5.1 Hz, 4H), 2.34 (s, 3H), 1.93-1.97 (m, 1H), 1.78-1.84 (m, 2H), 1.69-1.71 (m, 2H), 1.60 (m, 3H), 1.11-1.17 (m, 5H), 1.06 (t, J = 7.3 Hz, 3H).
13
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Chloroform-d) δ 170.64, 165.81, 157.41, 156.62, 150.00, 141.57, 112.36, 55.30, 47.35, 46.85, 45.99, 36.46, 33.32, 30.80, 26.29, 25.84, 23.13, 13.63. HR-MS (ESI): Calcd. C21H32N6OS, [M+H]+m/z: 417.2437, found: 417.2438.
4.2.17 8-Isobutyl-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (19) 18
ACCEPTED MANUSCRIPT Yellow waxy solid, yield 65%. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 4.17 (m, 4H), 4.03 (d, J = 3.6 Hz, 2H), 3.04 (t, J = 7.4 Hz, 2H), 2.44 (t, J = 5.0 Hz, 4H), 2.15-2.21 (m, 4H), 1.67-1.74 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H), 0.87 (d, J = 6.7 Hz, 6H).
13
C NMR (100 MHz, Chloroform-d) δ 174.65, 170.70, 157.39, 156.56, 149.96,
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141.69, 127.83, 125.77, 112.31, 55.01, 47.90, 47.03, 45.65, 33.29, 27.14, 23.05, 20.14, 13.60. HR-MS (ESI): Calcd. C18H28N6OS, [M+H]+m/z: 377.2124, found: 377.2125.
4.2.18 8-Benzyl-2-methyl-4-(4-methylpiperazin-1-yl)pteridin-7(8H)-one (20)
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Yellow solid, m. p. 142-143 oC, yield 61%. 1H NMR (400 MHz, Chloroform-d) δ 7.96 (s, 1H), 7.53-7.55 (m, 2H), 7.29 (m, 2H), 7.23 (m, 1H), 5.56 (s, 2H), 4.30 (m, 4H),
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2.52 (m, 7H), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.86, 157.55, 155.35, 149.54, 142.96, 136.41, 128.18, 127.99, 127.13, 111.94, 54.72, 46.82, 45.52, 43.20, 26.18. HR-MS (ESI): Calcd. C19H22N6O, [M+H]+m/z: 351.1933, found: 351.1932.
4.2.19 8-Benzyl-4-(4-methylpiperazin-1-yl)pteridin-7(8H)-one (21)
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White solid, m. p. 141-142 oC, yield 73%. 1H NMR (400 MHz, Chloroform-d) δ 8.39 (s, 1H), 8.04 (s, 1H), 7.50-7.52 (m, 2H), 7.29 (m,2H), 7.23 (m, 1H), 5.57 (s, 2H), 4.32 (m, 4H), 2.55 (m, 4H), 2.35 (s, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 157.62,
156.52, 155.30, 149.49, 144.21, 136.22, 128.22, 127.63, 127.12, 113.55, 54.69, 46.99,
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337.1777.
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45.48, 43.40. HR-MS (ESI): Calcd. C18H20N6O, [M+H]+m/z: 337.1777, found:
4.3 Antiproliferative activity assays Exponentially growing cells were seeded into 96-well plates at a concentration of 3,000 cells per well. After 24 h of incubation, the culture medium was removed and fresh medium containing various concentrations of the candidate compounds was added to each well. The cells were then incubated for 48 h, thereafter MTT assays were performed and cell viability was assessed at 570 nm by a microplate reader (Biotech, Shanghai, China). 19
ACCEPTED MANUSCRIPT 4.4 Hoechst 33258 staining MKN-45 cells were seeded into a 6-well plate (2×105/well) and incubated overnight for adherent and treated with compound 12 at different concentration for 24 h, and underwent Hoechst 33258 staining for 30 min in the dark. The cells were observed
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under a Nikon Eclipse TE 2000-S fluorescence microscope (Nikon, Japan).
4.5 Cell apoptosis assay
MKN-45 cells were seeded into a 6-well plate (2×105/well) and incubated for 24 h.
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Then the cells were treated with different concentrations of the tested compound 12 for 24 h. Thereafter, the cells were collected and the Annexin-V-FITC/PI apoptosis kit
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(Biovision) was used according to the manufacturer’s protocol. The cells were analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific, MA).
4.6 Western blot analysis
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MKN-45 cells were treated with different concentrations of compound 12 for 24 h, the cells were collected, lysed in RIPA buffer contained a protease inhibitor cocktail for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4 oC. After the
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collection of supernatant, the protein concentration was detected using a bicinchoninic acid assay kit (Beyotmie Biotechnology, Haimen, China). After added with loading buffer, cell lyses were boiled for 10 min at 100 oC for SDS- polyacrylamide gel
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electrophoresis (PAGE). Proteins were transferred to nitrocellulose (NC) membranes. Then the membranes were blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4o C with primary antibodies. After washing the membrane with the secondary antibody (1: 5000) at room temperature for 2 h. Finally, the blots were washed in TBST/TBS. The antibody-reactive were revealed by enhanced chemiluminescence (ECL) and exposed on Kodak radiographic film.
Acknowledgement This work was supported by National Key Research Programs of Proteins (No. 20
ACCEPTED MANUSCRIPT 2016YFA0501800); National Natural Science Foundation of China (Project No. 81430085, 81703326 and 21372206); Key Research Program of Henan Province (No. 1611003110100); the Starting Grant of Zhengzhou University (No. 32210533).
References pteridine derivatives, Res J. Pharm. Biol. Chem. Sci. 6 (2015) 899-904.
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[1] A. Amjed, Ai-Diksin, K. Hanoy, Ai-Amood, Syntheis and biological activity study of some new [2] G. Ferrand, H. Dumas, J.C. Depin, Y. Quentin, Synthesis and potential antiallergic activity of new pteridinones and related compounds, Eur. J. Med. Chem. 31 (1996) 273-280.
[3] Yoshitsugu Kokuryo, Takuji Nakatani, Makoto Kakinuma, Mikio Kabaki, Kyozo Kawata, Akira
M AN U
SC
Kugimiya, Kenji Kawada, Mitsunobu Matsumoto, Ryuji Suzuki, Mitsuaki Ohtani, New γ-fluoromethotrexates modified in the pteridine ring: synthesis and in vitro immunosuppressive activity, Eur. J. Med. Chem. 35 (2000) 529-534. [4] Eleni Pontiki, Dimitra Hadjipavlou-Litina, Alexandros Patsilinakos, Trang M Tran, Charles M Marson, Pteridine-2,4-diamine derivatives as radical scavengers and inhibitors of lipoxygenase that can possess anti-inflammatory properties, Future. Med. Chem. 7 (2015) 1937-1951. [5] A. Guirado, J.I. Lopez Sanchez, A.J. Ruiz-Alcaraz, P. Garcia-Penarrubia, D. Bautista, J. Galvez, First synthesis and biological evaluation of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines as inhibitors of TNF-alpha and IL-6, Eur. J. Med. Chem. 66 (2013) 269-275.
[6] G.A. Breault, J. Comita-Prevoir, C.J. Eyermann, B. Geng, R. Petrichko, P. Doig, E. Gorseth, B. Noonan, Exploring 8-benzyl pteridine-6,7-diones as inhibitors of glutamate racemase (MurI) in gram-positive
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bacteria, Bioorg. Med. Chem. Lett. 18 (2008) 6100-6103. [7] R.C. Reynolds, S. Srivastava, L.J. Ross, W.J. Suling, E.L. White, A new 2-carbamoyl pteridine that inhibits mycobacterial FtsZ, Bioorg. Med. Chem. Lett. 14 (2004) 3161-3164. [8] M.H. Li, S.K. Choi, T.P. Thomas, A. Desai, K.H. Lee, A. Kotlyar, M.M. Banaszak Holl, J.R. Baker, Jr., Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting,
EP
Eur. J. Med. Chem. 47 (2012) 560-572. [9] Z. Zhang, J. Wu, F. Ran, Y. Guo, R. Tian, S. Zhou, X. Wang, Z. Liu, L. Zhang, J. Cui, J. Liu, Novel 8-deaza-5,6,7,8-tetrahydroaminopterin derivatives as dihydrofolate inhibitor: design, synthesis and
AC C
antifolate activity, Eur. J. Med. Chem. 44 (2009) 764-771. [10] S.A. Langie, S. Achterfeldt, J.P. Gorniak, K.J. Halley-Hogg, D. Oxley, F.J. van Schooten, R.W. Godschalk, J.A. McKay, J.C. Mathers, Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring, Faseb J. 27 (2013) 3323-3334. [11] K.K. Liu, S. Bagrodia, S. Bailey, H. Cheng, H. Chen, L. Gao, S. Greasley, J.E. Hoffman, Q. Hu, T.O. Johnson, D. Knighton, Z. Liu, M.A. Marx, M.D. Nambu, S. Ninkovic, B. Pascual, K. Rafidi, C.M. Rodgers, G.L. Smith, S. Sun, H. Wang, A. Yang, J. Yuan, A. Zou, 4-methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors, Bioorg. Med. Chem. Lett. 20 (2010) 6096-6099. [12] W. Zhou, X. Liu, Z. Tu, L. Zhang, X. Ku, F. Bai, Z. Zhao, Y. Xu, K. Ding, H. Li, Discovery of pteridin-7(8H)-one-based irreversible inhibitors targeting the epidermal growth factor receptor (EGFR) kinase T790M/L858R mutant, J. Med. Chem. 56 (2013) 7821-7837. [13] P.A. Roethle, R.M. McFadden, H. Yang, P. Hrvatin, H. Hui, M. Graupe, B. Gallagher, J. Chao, J. Hesselgesser, P. Duatschek, J. Zheng, B. Lu, D.B. Tumas, J. Perry, R.L. Halcomb, Identification and 21
ACCEPTED MANUSCRIPT optimization of pteridinone Toll-like receptor 7 (TLR7) agonists for the oral treatment of viral hepatitis, J. Med. Chem. 56 (2013) 7324-7333. [14] Z.H. Li, X.Q. Liu, P.F. Geng, F.Z. Suo, J.L. Ma, B. Yu, T.Q. Zhao, Z.Q. Zhou, C.X. Huang, Y.C. Zheng, H.M. Liu, Discovery of [1,2,3]Triazolo[4,5-d]pyrimidine Derivatives as Novel LSD1 Inhibitors, ACS Med. Chem. Lett. 8 (2017) 384-389. [15] Z.H. Li, D.X. Yang, P.F. Geng, J. Zhang, H.M. Wei, B. Hu, Q. Guo, X.H. Zhang, W.G. Guo, B. Zhao, B. Yu, L.Y. Ma, H.M. Liu, Design, synthesis and biological evaluation of [1,2,3]triazolo[4,5-d]pyrimidine
RI PT
derivatives possessing a hydrazone moiety as antiproliferative agents, Eur. J. Med. Chem. 124 (2016) 967-980.
[16] Z.H. Li, J. Zhang, X.Q. Liu, P.F. Geng, J.L. Ma, B. Wang, T.Q. Zhao, B. Zhao, H.M. Wei, C. Wang, D.J. Fu, B. Yu, H.M. Liu, Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy, Eur. J. Med. Chem. 135 (2017) 204-212.
[17] S. Wang, L.J. Zhao, Y.C. Zheng, D.D. Shen, E.F. Miao, X.P. Qiao, L.J. Zhao, Y. Liu, R. Huang, B. Yu, H.M.
SC
Liu, Design, synthesis and biological evaluation of [1,2,4]triazolo[1,5-a]pyrimidines as potent lysine specific demethylase 1 (LSD1/KDM1A) inhibitors, Eur. J. Med. Chem. 125 (2017) 940-951. [18] Y.C. Duan, Y.C. Ma, E. Zhang, X.J. Shi, M.M. Wang, X.W. Ye, H.M. Liu, Design and synthesis of novel
M AN U
1,2,3-triazole-dithiocarbamate hybrids as potential anticancer agents, Eur. J. Med. Chem. 62 (2013) 11-19.
[19] R.S. Harapanhalli, R.W. Howell, D.V. Rao, Bis-benzimidazole dyes, Hoechst 33258 and Hoechst 33342: radioiodination, facile purification and subcellular distribution, Nucl. Med. Biol. 21 (1994) 641-647.
[20] Z. Chen, X. Liang, H.Y. Zhang, H. Xie, J.W. Liu, Y.F. Xu, W.P. Zhu, Y. Wang, X. Wang, S.Y. Tan, D. Kuang, X.H. Qian, A New Class of Naphthalimide-Based Antitumor Agents That Inhibit Topoisomerase II and
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Induce Lysosomal Membrane Permeabilization and Apoptosis, J. Med. Chem. 53 (2010) 2589-2600. [21] X.J. Shi, B. Yu, J.W. Wang, P.P. Qi, K. Tang, X. Huang, H.M. Liu, Structurally novel steroidal spirooxindole by241 potently inhibits tumor growth mainly through ROS-mediated mechanisms, Sci.
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Rep. 6 (2016) 31607.
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ACCEPTED MANUSCRIPT
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Highlights The pteridin-7(8H)-one derivatives showed potent inhibition against the cancer cells. Compound 12 exerted the most potent and broad-spectrum antiproliferative activity. Compound 12 induced the apoptosis and G2/M arrest of MKN-45 cells. A novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold was constructed.
S0223-5234(17)30837-1
DOI:
10.1016/j.ejmech.2017.10.037
Reference:
EJMECH 9829
To appear in:
European Journal of Medicinal Chemistry
Received Date: 22 June 2017 Revised Date:
11 October 2017
Accepted Date: 13 October 2017
Please cite this article as: Z.-H. Li, T.-Q. Zhao, X.-Q. Liu, B. Zhao, C. Wang, P.-F. Geng, Y.-Q. Cao, D.-J. Fu, L.-P. Jiang, B. Yu, H.-M. Liu, Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.10.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Graphic abstract
Pteridin-7(8H)-ones were synthesized and evaluated for their antiproliferative activity.
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Compound 12 displayed acceptable growth inhibition, induced apoptosis of MKN-45 cells. Besides, a novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold was first prepared.
ACCEPTED MANUSCRIPT Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives Zhong-Hua Li a, b, c, d, 1,Tao-Qian Zhao a, b, c, d, 1, Xue-Qi Liu a, b, c, d, Bing Zhao a, b, c, d, Chao Wang a, b, c, d, Peng-Fei Geng a, b, c, d, Ya-Quan Cao a, b, c, d, Dong-Jun Fu a, b, c, d,
a
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Li-Ping Jiang e, Bin Yu a, b, c, d,*, Hong-Min Liu a, b, c, d,* Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan
Province;
b
Key Laboratory of Technology of Drug Preparation (Zhengzhou University),
Ministry of Education of China; c Key Laboratory of Henan Province for Drug Quality and
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Evaluation; d School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China; e School of Basic Medical Science，Central South University, Changsha 410013,
*Corresponding Authors: Prof. Hong-Min Liu & Dr. Bin Yu
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PR China
Zhengzhou University, Zhengzhou, 450001, China
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E-mail: [email protected] (Hong-Min Liu) & [email protected] (Bin Yu)
ABSTRACT: Pteridines are an important class of heterocyclic compounds with diverse biological activities. Here, we report a series of pteridin-7(8H)-one derivatives
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and their antiproliferative activities toward MKN-45, MGC-803, EC-109, and H1650. Structure-activity relationship studies showed that compound 12 exerted the most
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potent antiproliferative activity against MKN-45 and MGC-803 with the IC50 values of 4.32 and 7.01 µM, respectively. Besides, compound 12 induced morphological changes and apoptosis of MKN-45 cells, increased expression of Bax, down-regulated expression of Bcl-2 and caused cleavage of caspase-3/9. Additionally, we first reported the construction of the novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and a chiral carbon center established. Keywords: Pteridin-7(8H)-one, Antiproliferative activity, Apoptosis, Cell cycle arrest
1
ACCEPTED MANUSCRIPT 1. Introduction Pteridines are an important class of bicyclic N-heteroarenes, which have received considerable attention from medicinal community due to their diverse biological activities, such as antimicrobial [1], antiallergic [2], immunosuppressive [3],
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anti-inflammatory [4, 5], antibacterial [6, 7], and anticancer activities [8, 9]. Some biologically active natural products (e.g. folic acid, etc.) are also found to possess pteridine scaffolds. As shown in Fig. 1, folic acid (also named as vitamin B9 or vitamin M) is necessary for human health and is involved in DNA synthesis, DNA
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repair and DNA methylation [10]. Antifolate methotrexate (MTX) is a chemotherapy agent and immune system suppressant for treatment of cancer and autoimmune
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diseases . Moreover, pteridine analogs are well known to act upon a wide range of targets for therapeutic potential [4]. Compound A, as a selective PI3K/mTOR dual inhibitor, is highly efficacious in mouse xenograft model bearing glioma cell line U87 [11]. Compound B has been proved to be able to inhibit EGFRwt and EGFRT790M/L858R potently and exerts remarkable in vivo growth inhibition of human non-small cell lung
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cancers (NSCLC) [12]. Compound C is a pteridinone-based TLR7 (Toll-like receptor 7) agonist, which is currently in clinical evaluation for the treatment of chronic HBV (hepatitis B) infection [13] (Fig. 1). The diverse bioactivities and natural prevalence
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make pteridines and their structural analogs promising bicyclic scaffolds to develop new agents for the treatment of different diseases.
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Following our previous work on the synthesis of new bicyclic heterocycles with anticancer potentials [14-17], we herein report the synthesis of a series of new pteridin-7(8H)-one derivatives through cascade reactions, their antiproliferative activity and preliminary mechanisms of inducing cancer cell death. Also, we first report the synthesis of new 8,9-dihydro-7H-purine-8-carboxylate via the 5-endo cyclization.
2
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2. Results and discussion 2.1 Chemistry
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Fig. 1. Biologically active pteridine derivatives.
The general synthetic route is illustrated in Scheme 1. The intermediate derivatives 2a-h were synthesized by condensation of commercially available pyrimidine analogs
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1a-c with appropriate primary amines in the presence of N,N-diisopropylethylamine (DIPEA) in DMF. The obtained intermediates 2a-h reacted with ethyl glyoxalate in AcOH/EtOH under heating to afford pteridin-7(8H)-one skeletons 3a-h [11], which
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then reacted with various amines in the presence of TEA in a mixed solvent of ethanol
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and DMF, generating the target compounds 4-21.
3
ACCEPTED MANUSCRIPT O Cl
Cl NH2
N R1
R2CH2NH2
1a-c
NH2
N
DMF, DIPEA, 100 oC, 4 h
Cl
N
N
R1
H
AcOH/ethanol (1 : 1) 100 oC, 2h
N
N
O R2
N H
Cl OEt
N
N
R1
3a-h
2a-h
1a, R1 = propyl-S1b, R1 = Me1c, R1 = H-
O R2
2a-3a, R1 = propyl-S-, R2 =Ph2b-3b, R1 = propyl-S-, R2 =PhC2H4-
N
1
R
Ethanol/DMF (3 : 1) reflux, 3h
2e-3e, R1 = propyl-S-, R2 =
N
N
R'RNH/TEA
2d-3d, R1 = H-, R2 =Ph-
R'
2f-3f, R1 = propyl-S-, R2 = N 4-21
N
S
O
2g-3g, R1 = propyl-S-, R2 =
R2
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R
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2c-3c, R1 = Me-, R2 =Ph-
O
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2h-3h, R1 = propyl-S-, R2 =
Scheme 1. Synthesis of pteridin-7(8H)-one derivatives.
Interestingly, compound 3h' was also observed in the conversion from compound 2h to
compound
3h.
Compound
3h'
features
a
novel
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8,9-dihydro-7H-purine-8-carboxylate scaffold. Both compounds were formed through the unstable imine intermediate A. For the synthesis of compound 3h, it involved the 6-exo cyclization (Scheme 2, path b), generating one C-N bond, one C=N bond, and the pteridin-7(8H)-onescaffold. While compound 3h' was competitively formed via
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the 5-endo cyclization (Scheme 2, path a), accompanying by the formation of two C-N bonds, a chiral center, and the 8,9-dihydro-7H-purine-8-carboxylate architecture.
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To the best of our knowledge, this is the first report on the rapid construction of the new
8,9-dihydro-7H-purine-8-carboxylate
framework,
which
also
possesses
pluripotent groups (e.g. –COOEt, Cl) for further structural derivations. Besides, the C-N single bond of compound 3h' could be potentially oxidized to aromatic bicyclic N-heteroarene.
4
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Scheme 2. Proposed reaction mechanism for the synthesis of compounds 3h and 3h'.
Compound 3h' was then characterized by 2D NMR spectra (see supporting
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information for details). Intriguingly, the methylene protons (highlighted in red) appeared at 3.99 and 3.17 ppm, respectively. The unique intramolecular H-bond interactions and the adjacent chiral center may be responsible for the remarkably different chemical shifts of the methylene protons.
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2.2 Evaluation of biological activity 2.2.1 Antiproliferative activity
Compounds 4-21 were evaluated for their antiproliferative activity against MKN-45
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and MGC803 (human gastric cancer cell lines), EC-109 (human esophageal cancer cell line), and H1650 (human lung cancer cell line), by using the MTT assay, and
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5-fluorouracil (5-FU) was employed as the reference drug [18]. The preliminary data are summarized in Tables 1-3.
As shown in Table 1, compounds 4-6 with different aniline substituents exerted moderate antiproliferative activity against the tested cancer cell lines, the substituents attached had certain effect on the activity, especially for MKN-45 and MGC-803 cells. Compound 6 with the 3,4,5-trimethoxyl group was more potent than compound 4 with the IC50 values of 14.25 and 15.03 µM, respectively against MKN-45 and MGC-803 cells. Compounds 7, 8, 13 and 14 with the aliphatic amine substituents showed 5
ACCEPTED MANUSCRIPT decreased inhibitory activity against the tested cancer cell lines. Compound 12 was significantly more potent than compounds 9-10, highlighting the importance of nitrogen atom of cyclic amine groups for the activity. Compound 11 with a benzyl group attached to the piperazine nitrogen atom showed decreased activity compared
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to compound 12. Compound 12 displayed good and broad-spectrum antiproliferative activities with IC50 values less than 10 µM, comparable to that of 5-FU.
Table 1
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Inhibitory activity of compounds 4-14 against the tested cancer cell lines
IC50 (µM)a
Compound
R'RN-
MGC-803
EC-109
H1650
38.60±3.75
36.60±4.69
19.03±1.38
>64
14.65±1.80
19.35±0.56
>64
51.84±3.86
14.25±1.42
15.03±1.86
38.67±1.56
36.94±0.96
7
>64
46.64±2.56
>64
>64
8
21.02±1.16
30.40±2.54
36.04±0.98
>64
9
>64
49.64±0.86
>64
>64
10
>64
34.71±1.43
>64
54.80±2.43
11
15.43±2.47
18.71±5.69
>64
>64
5
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6
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4
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MKN-45
6
ACCEPTED MANUSCRIPT 12
4.32±1.87
7.01±3.61
9.85±2.81
9.92±1.41
13
39.23±4.72
>64
>64
>64
14
23.26±1.86
43.74±1.80
29.71±1.65
>64
8.89±1.65
7.52±0.98
6.21±0.75
14.25±2.73
a
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-
5-Fu
Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs
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of three independent experiments.
Next, the influence of R2 substituents on the activity was also investigated. As shown
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in Table 2, compounds 15-19 generally displayed decreased or comparable activities toward the tested cancer cells, regardless of the patterns of R2 group.
Table 2
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Inhibitory activity of compounds 15-19 against the tested cancer cell lines
MKN-45
MGC-803
EC-109
H1650
15
9.97±0.97
13.75±3.81
18.28±2.95
>64
16
11.32±1.57
16.41±3.16
19.69±6.07
>64
17
11.56±6.50
>64
24.04±4.88
54.91±6.08
Compound
R
IC50 (µM)a
2
7
ACCEPTED MANUSCRIPT 18
17.92±2.82
53.67±5.66
>64
51.81±3.21
19
16.83±5.65
17.17±2.35
37.48±4.62
45.43±2.90
8.89±1.65
7.52±0.98
6.21±0.75
14.25±2.73
5-Fu
Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
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a
-
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent experiments.
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However, the replacement of propylthio group with methyl (compound 20) or hydrogen (compound 21) led to the loss of antiproliferative activity against the tested
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cancer cell lines, significantly less potent than 5-FU and compound 12 (Table 3).
Table 3
R
a
IC50 (µM)a
1
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Compound
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Inhibitory activity of compounds 20-21 against the tested cancer cell lines
MKN-45
MGC-803
EC-109
H1650
12
propyl-S-
4.32±1.87
7.01±3.61
9.85±2.81
24.91±4.41
20
Me-
>64
>64
38.98±0.99
>64
21
H
>64
>64
41.66±5.02
>64
5-Fu
-
8.89±1.65
7.52±0.98
6.21±0.75
14.25±2.73
Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent experiments. 8
ACCEPTED MANUSCRIPT
2.2.2 Cell apoptosis of MKN-45 and possible mechanism involved The acceptable antiproliferative activity of compound 12 promoted us to investigate its effect on the apoptosis and morphological changes. Hochest 33258 staining was
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performed to investigate morphological changes of MKN-45 cells [19]. After 24 h incubation with compound 12 at indicated concentrations, characteristic apoptotic morphological changes were observed, including cell rounding, chromatin shrinkage and formation of apoptotic bodies (Fig. 2A). Notably, this phenomena were more
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remarkable at higher concentrations. To further explore the effect of compound 12 on cell apoptosis, the apoptotic analysis was also performed with Annexin V-FITC/PI
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double staining and analyzed with flow-cytometry calculation [20]. Treatment of MKN-45 cells with compound 12 resulted in a concentration-dependent apoptosis
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increase (Fig. 2B/2C).
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Fig. 2. Compound 12 induced apoptosis of MKN-45 cells. (A) Apoptosis analysis with Hoechst
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33258 staining; (B) Apoptosis effect on MKN-45 cell line induced by compound 12 for 24 h using Annexin V-FITC/PI double staining and flow-cytometry calculation. The lower left quadrant
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represents live cells, the lower right is for early/primary apoptotic cells, upper right is for late/secondary apoptotic cells, and the upper left represents cells damaged during the procedure; (C) Quantitative analysis of apoptotic cells. The experiments were performed three times, and a representative experiment is shown.
Next, the western blot analysis was performed to examine the expression of apoptosis-related proteins. As shown in Fig. 3A, treatment of MKN-45 cells with compound 12 increased expression of Bax in a concentration-dependent manner (Fig. 3B). Bax was able to activate the caspases, and promoted the release of cytochrome c 10
ACCEPTED MANUSCRIPT and other pro-apoptotic factors from the mitochondria [21]. Meanwhile, the expression of anti-apoptotic protein Bcl-2 decreased accordingly (Fig. 3C). As shown in Fig. 3D and 3E, treatment of compound 12 also concentration-dependently caused the cleavage of caspases-3 and caspases-9, which then inducing cell apoptosis. These
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results indicated that compound 12 may induce MKN-45 apoptosis through the
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intrinsic apoptotic pathway.
Fig. 3. Expression changes of apoptosis-related proteins induced by compound 12. (A) Compound 12 induced expression changes of Bax, Bcl-2 and caspase family members in MKN-45 cells; (B-E) Statistical analysis of expression levels of Bax, Bcl-2 and cleaved-caspased 9/3.
2.2.3 Cell cycle analysis The effect of compound 12 on the cell cycle was also evaluated. After treatment of MKN-45 cells with compound 12 for 24 h at indicated concentrations (0, 2, 4, 8 µM), 11
ACCEPTED MANUSCRIPT the percentage of cells in G2/M phase were 1.62%, 16.6%, 29.2% and 40.8%, respectively (Fig. 4), suggesting that compound 12 caused an obvious G2/M arrest in a concentration-dependent manner with concomitant decrease in terms of the number
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of cells in other phases of the cell cycle.
Fig. 4. Effect of compound 12 on the cell cycle distribution of MKN-45 cells. Cells were treated with indicated concentrations of compound 12 (0, 2, 4, 8 µM) for 24 h. Then the cells were fixed and stained with PI to analyze DNA content by flow cytometry. The experiments were performed three times and a representative experiment is shown.
3. Conclusions In summary, a new series of pteridin-7(8H)-one derivatives were prepared and evaluated for their antiproliferative activity. Among them, compound 12 exhibited the 12
ACCEPTED MANUSCRIPT most potent and broad-spectrum inhibition against the tested cancer cells (MKN-45, MGC803, EC-109 and H1650) and displayed comparable activity with the anticancer drug 5-FU. Besides, compound 12 induced morphological changes and apoptosis of MKN-45 cells in a concentration-dependent manner, increased expression of
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pro-apoptotic protein Bax, down-regulated expression of anti-apoptotic protein Bcl-2 and caused cleavage of caspase-3/9, indicating that compound 12 induced apoptosis via the intrinsic apoptotic pathway. The G2/M phase arrest induced by compound 12 was also observed. Additionally, we first reported the construction of the novel
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bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and a chiral carbon center established.
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This scaffold contains several pluripotent handles, which allows for further structural modifications for identifying potential bioactive molecules.
4. Experimental section 4.1 General
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Reagents and solvents were purchased from commercial sources and were used without further purification. Melting points were determined on an X-5 micromelting apparatus and are uncorrected. 1H NMR and
13
C NMR spectra were recorded on a
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Bruker (DPX-400) spectrometer, operating at 400 and 100 MHz, respectively. High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-Tof of
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Micromass spectrometer by electrospray ionizaton (ESI).
4.2 General procedure for the synthesis of compounds 3h', 4-21. A solution of 2a (1eq) with ethyl glyoxalate (50 wt. % in toluene, 1.2 eq) in a mixed solvent of AcOH/ethanol (1 : 1) was heated to 100 oC for 2 h. After the completion of the reaction monitored by TLC (PE/EA = 4 : 1), the purification was conducted by flash column chromatography on silica gel using PE/EA (4 : 1) as eluent to afford 3a. As a representative example, the new compound 3h' was separated and characterized by NMR. Compounds 4 can be readily obtained by refluxing 3a (1eq), aniline (1.2 eq) and triethylamine (1.2 eq) in the mixed solvent of ethanol/DMF (3 : 1) for 3 h, 13
ACCEPTED MANUSCRIPT following by purification on flash column chromatography on silica gel. This procedure was also applied to preparation of compounds 5-21.
4.2.1 Ethyl (R)-6-chloro-9-(cyclohexylmethyl)-2-(propylthio)-8,9-dihydro-7H-purine-
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8-carboxylate (3h') Pale yellow solid, yield 40%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 5.10 (s, 1H), 3.96-4.01 (m, 1H), 3.60-3.66 (m, 2H), 3.5-3.20 (m, 1H), 3.04-3.11 (m, 1H), 2.93-3.00 (m, 1H), 1.61-1.73 (m, 8H), 1.14-1.20 (m, 3H), 1.09 (t, J = 7.0 Hz, 3H),
SC
0.98 (t, J = 7.3 Hz, 3H), 0.90-0.96 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.3, 160.2, 150.0, 141.1, 112.2, 85.9, 64.4, 52.2, 36.1, 32.3, 30.1, 30.1, 26.0, 25.3, 25.3,
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22.6, 14.9, 13.3. HR-MS (ESI): Calcd. C18H27ClN4O2S, [M+H]+m/z: 399.1621, found: 399.1623.
4.2.2 8-Benzyl-4-(phenylamino)-2-(propylthio)pteridin-7(8H)-one (4) Pink solid, yield 75%. 1H NMR (400 MHz, Chloroform-d) δ 8.42 (s, 1H), 7.98 (s, 1H),
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7.75 (m, 1H), 7.49-7.51 (m, 2H), 7.36-7.40 (m, 2H), 7.23-7.34 (m, 3H), 7.12-7.16 (m, 1H), 5.51 (s, 2H), 3.13 (t, J = 7.2 Hz, 2H), 1.73-1.83 (m, 2H), 1.04 (t, J = 7.3 Hz, 3H). 13
C NMR (100 MHz, Chloroform-d) δ 173.50, 156.99, 155.61, 147.37, 144.76,
137.85, 135.89, 129.01, 128.51, 127.85, 124.11, 120.41, 110.37, 43.77, 33.43, 22.99,
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13.61.HR-MS (ESI): Calcd. C22H21N5OS, [M+K]+m/z: 442.1104, found: 442.1102.
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4.2.3 8-Benzyl-4-((2-methoxyphenyl)amino)-2-(propylthio)pteridin-7(8H)-one (5) Yellow solid, yield 81%. 1H NMR (400 MHz, Chloroform-d) δ 9.15 (s, 1H), 8.62-8.64 (m, 1H), 8.02 (s, 1H), 7.49 (m, 2H), 7.27-7.33 (m, 2H), 6.93-7.10 (m, 3H), 5.52 (s, 2H), 3.97 (s, 3H), 3.15 (t, J = 7.4 Hz, 2H), 1.75-1.83 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H).
13
C NMR (100 MHz, Chloroform-d) δ 173.36, 157.03, 155.39, 148.79,
147.26, 144.77, 135.96, 128.97, 128.49, 127.80, 127.67, 123.54, 120.78, 119.99, 110.99, 110.12, 55.90, 43.74, 33.43, 23.00, 13.63.HR-MS (ESI): Calcd. C23H23N5O2S, [M+Na]+m/z: 456.1470, found: 456.1472. 14
ACCEPTED MANUSCRIPT 4.2.4
8-Benzyl-2-(propylthio)-4-((3,4,5-trimethoxyphenyl)amino)pteridin-7(8H)-one
(6) Yellow solid, yield 86%. 1H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.99 (s, 1H), 7.47-7.49 (m, 2H), 7.28-7.32 (m, 3H), 7.07 (s, 2H), 5.53 (s, 2H), 3.90 (s, 6H),
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3.85 (s, 3H), 3.15 (t, J = 7.3 Hz, 2H), 1.72-1.77 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 173.46, 156.97, 155.48, 153.36, 147.30, 144.76, 135.84, 134.77, 133.88, 128.80, 128.52, 127.85, 110.35, 98.32, 61.02, 56.21, 43.83, 33.40, 22.56, 13.49. HR-MS (ESI): Calcd. C25H27N5O4S, [M+Na]+m/z: 516.1681,
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found: 516.1682.
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4.2.5 8-Benzyl-4-(benzylamino)-2-(propylthio)pteridin-7(8H)-one (7)
Yellow solid, yield 68%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.48 (m, 2H), 7.35 (m, 4H), 7.26-7.31 (m, 4H), 6.78 (t, J = 5.6 Hz, 1H), 5.49 (s, 2H), 4.77 (d, J = 5.9 Hz, 2H), 3.09 (t, J = 7.3 Hz, 2H), 1.69-1.78 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13
C NMR (100 MHz, Chloroform-d) δ 173.22, 158.01, 157.14, 147.19, 144.21,
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137.88, 136.05, 128.92, 128.78, 128.46, 127.76, 127.70, 110.33, 44.80, 43.75, 33.39, 22.88, 13.58. HR-MS (ESI): Calcd. C23H23N5OS, [M+Na]+m/z: 440.1521, found:
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440.1524.
4.2.6 8-Benzyl-4-((cyclohexylmethyl)amino)-2-(propylthio)pteridin-7(8H)-one (8) White solid, yield 76%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.46-7.49
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(m, 2H), 7.27-7.31 (m, 2H), 7.22-7.25 (m, 1H), 6.57 (t, J = 6.1 Hz, 1H), 5.48 (s, 2H), 3.42 (t, J = 6.5 Hz, 2H), 3.09 (t, J = 7.4 Hz, 2H), 1.62-1.80 (m, 8H), 1.16-1.30 (m, 3H), 0.96-1.05 (m, 5H).
13
C NMR (100 MHz, Chloroform-d) δ 173.05, 158.35,
157.20, 147.03, 143.84, 136.12, 128.94, 128.43, 127.71, 110.35, 47.04, 43.69, 37.99, 33.37, 30.88, 26.37, 25.83, 23.01, 13.62. HR-MS (ESI): Calcd. C23H29N5OS, [M+Na]+m/z: 446.1991, found: 446.1992.
4.2.7 8-Benzyl-4-morpholino-2-(propylthio)pteridin-7(8H)-one (9) 15
ACCEPTED MANUSCRIPT Yellow solid, yield 79%. 1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.45 (m, 2H), 7.29 (m, 2H), 7.23 (m, 1H), 5.53 (s, 2H), 4.27-4.30 (m, 4H), 3.80 (t, J = 4.7 Hz, 4H), 3.05 (t, J = 7.3 Hz, 2H), 1.68-1.77 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 171.09, 157.27, 156.17, 149.70, 141.83, 136.15, 128.73,
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128.43, 127.66, 112.36, 67.09, 47.94, 44.09, 33.29, 22.85, 13.56. HR-MS (ESI): Calcd. C20H24N5O2S, [M+Na]+m/z: 420.1470, found: 420.1471.
4.2.8 8-Benzyl-2-(propylthio)-4-thiomorpholinopteridin-7(8H)-one (10)
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White solid, yield 81%. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.46 (m, 2H), 7.24-7.31 (m, 3H), 5.52 (s, 2H), 4.49 (t, J = 4.9 Hz, 4H), 3.05 (t, J = 7.3 Hz, 2H), 13
C NMR (100
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2.76 (t, J = 5.0 Hz, 4H), 1.70-1.76 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).
MHz, Chloroform-d) δ 171.06, 157.23, 156.13, 149.80, 141.94, 136.17, 128.82, 128.42, 127.68, 112.34, 50.70, 44.09, 33.31, 27.62, 22.91, 13.58. HR-MS (ESI): Calcd. C20H23N5OS2, [M+Na]+m/z: 436.1242, found: 436.1245.
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4.2.9 8-Benzyl-4-(4-benzylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (11) White solid, yield 72%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.45 (m, 2H), 7.23-7.34 (m, 8H), 5.51 (s, 2H), 4.28 (m, 4H), 3.54 (s, 2H), 3.04 (t, J = 7.3 Hz,
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2H), 2.56 (t, J = 5.0 Hz, 4H), 1.67-1.76 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.88, 157.15, 156.22, 149.68, 141.48, 137.60, 136.25, 129.21, 128.79, 128.40, 128.33, 127.62, 127.27, 112.35, 62.96, 53.31, 47.44, 44.04,
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33.28, 22.89, 13.56. HR-MS (ESI): Calcd. C27H30N6OS, [M+H]+m/z: 487.2280, found:487.2281.
4.2.10 8-Benzyl-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (12) White solid, yield 82%. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.44-7.46 (m, 2H), 7.28-7.31 (m, 1H), 7.22-7.26 (m, 2H), 5.52 (s, 2H), 4.30 (m, 4H), 3.05 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 4.9 Hz, 4H), 2.34 (s, 3H), 1.68-1.77 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.64, 156.60, 155.33, 149.36, 141.68, 16
ACCEPTED MANUSCRIPT 136.17, 128.19, 127.33, 127.06, 111.54, 54.65, 46.89, 45.50, 43.49, 32.45, 22.48, 13.18. HR-MS (ESI): Calcd. C21H26N6OS, [M+H]+m/z: 411.1967, found: 411.1968.
4.2.11 8-Benzyl-4-(propylamino)-2-(propylthio)pteridin-7(8H)-one (13)
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Yellow solid, yield 85%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 6.0 Hz, 1H), 7.96 (s, 1H), 7.24-7.31 (m, 5H), 5.35 (s, 2H), 3.38-3.43 (m, 2H), 2.99 (t, J = 7.2 Hz, 2H), 1.54-1.66 (m, 2H), 0.86-0.92 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.85, 153.02, 151.93, 141.78, 138.66, 130.86, 123.64, 123.17, 122.44, 105.10, 38.44, 37.37,
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28.12, 17.71, 17.54, 8.32, 6.16. HR-MS (ESI): Calcd. C19H23N5OS, [M+Na]+m/z:
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392.1521, found:392.1522.
4.2.12 8-Benzyl-4-(cyclopropylamino)-2-(propylthio)pteridin-7(8H)-one (14) Yellow solid, yield 68%. 1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 4.5 Hz, 1H), 7.95 (s, 1H), 7.22-7.32 (m, 5H), 5.36 (s, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.96 (m, 1H), 1.58-1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H), 0.68-0.74 (m, 4H). 13C NMR (100 MHz,
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DMSO-d6) δ 171.37, 158.96, 156.27, 146.85, 143.68, 136.24, 128.30, 127.30, 127.13, 110.00, 43.14, 32.51, 24.04, 22.63, 13.24, 5.98. HR-MS (ESI): Calcd. C19H21N5OS,
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[M+Na]+m/z: 390.1365, found: 390.1366.
4.2.13 4-(4-Methylpiperazin-1-yl)-8-(3-phenylpropyl)-2-(propylthio)pteridin-7(8H)one (15)
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Yellow solid, yield 63%. 1H NMR (400 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.28 (m, 1H), 7.25 (m, 1H), 7.15-7.21 (m, 3H), 4.35-4.39 (m, 2H), 4.31-4.32 (m, 4H), 3.03 (t, J = 7.2 Hz, 2H), 2.73 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 5.1 Hz, 4H), 2.36 (s, 3H), 2.03-2.08 (m, 2H), 1.74-1.79 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 170.96, 157.26, 156.20, 149.64, 141.53, 141.08, 128.35, 128.23, 125.95, 112.34, 54.96, 46.94, 45.58, 41.08, 33.34, 33.32, 28.72, 22.92, 13.56. HR-MS (ESI): Calcd. C23H30N6OS, [M+H]+m/z: 439.2280, found: 439.2281.
4.2.14 4-(4-Methylpiperazin-1-yl)-2-(propylthio)-8-(thiophen-2-ylmethyl)pteridin17
ACCEPTED MANUSCRIPT 7(8H)-one (16) Yellow waxy solid, yield 78%. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.34-7.36 (m, 1H), 7.14 (d, J = 3.5 Hz, 1H), 6.92-6.94 (m, 1H), 5.53 (s, 2H), 4.21-4.23 (m, 4H), 3.12 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 5.0 Hz, 4H), 2.23 (s, 3H), 13
C NMR (100 MHz, Chloroform-d) δ
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1.70-1.77 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H).
170.97, 157.15, 155.63, 149.19, 141.53, 137.29, 128.87, 126.39, 126.10, 112.28, 55.14, 47.15, 45.81, 38.42, 33.34, 22.88, 13.62. HR-MS (ESI): Calcd. C19H24N6OS2,
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[M+H]+m/z: 417.1531, found: 417.1532.
4.2.15 8-(Furan-2-ylmethyl)-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-
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one (17)
Yellow waxy solid, yield 73%. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.55 (d, J = 1.7 Hz, 1H), 6.39 (m, 1H), 6.29 (d, J = 3.2 Hz, 1H), 5.38 (s, 2H), 4.18 (m, 4H), 3.04 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 5.0 Hz, 4H), 2.20 (s, 3H), 1.63-1.73 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, Chloroform-d) δ 171.04, 157.20, 155.80,
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149.37, 142.06, 141.56, 112.27, 110.41, 109.42, 55.06, 47.08, 45.72, 37.04, 33.33,
401.1762.
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22.94, 13.58. HR-MS (ESI): Calcd. C19H24N6O2S, [M+H]+m/z: 401.1760, found:
4.2.16 8-(Cyclohexylmethyl)-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)one (18)
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White solid, m. p. 146-147 oC, yield 72%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 4.29 (m, 4H), 4.19 (d, J = 7.3 Hz, 2H), 3.07 (t, J = 7.4 Hz, 2H), 2.53 (t, J = 5.1 Hz, 4H), 2.34 (s, 3H), 1.93-1.97 (m, 1H), 1.78-1.84 (m, 2H), 1.69-1.71 (m, 2H), 1.60 (m, 3H), 1.11-1.17 (m, 5H), 1.06 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz,
Chloroform-d) δ 170.64, 165.81, 157.41, 156.62, 150.00, 141.57, 112.36, 55.30, 47.35, 46.85, 45.99, 36.46, 33.32, 30.80, 26.29, 25.84, 23.13, 13.63. HR-MS (ESI): Calcd. C21H32N6OS, [M+H]+m/z: 417.2437, found: 417.2438.
4.2.17 8-Isobutyl-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (19) 18
ACCEPTED MANUSCRIPT Yellow waxy solid, yield 65%. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 4.17 (m, 4H), 4.03 (d, J = 3.6 Hz, 2H), 3.04 (t, J = 7.4 Hz, 2H), 2.44 (t, J = 5.0 Hz, 4H), 2.15-2.21 (m, 4H), 1.67-1.74 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H), 0.87 (d, J = 6.7 Hz, 6H).
13
C NMR (100 MHz, Chloroform-d) δ 174.65, 170.70, 157.39, 156.56, 149.96,
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141.69, 127.83, 125.77, 112.31, 55.01, 47.90, 47.03, 45.65, 33.29, 27.14, 23.05, 20.14, 13.60. HR-MS (ESI): Calcd. C18H28N6OS, [M+H]+m/z: 377.2124, found: 377.2125.
4.2.18 8-Benzyl-2-methyl-4-(4-methylpiperazin-1-yl)pteridin-7(8H)-one (20)
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Yellow solid, m. p. 142-143 oC, yield 61%. 1H NMR (400 MHz, Chloroform-d) δ 7.96 (s, 1H), 7.53-7.55 (m, 2H), 7.29 (m, 2H), 7.23 (m, 1H), 5.56 (s, 2H), 4.30 (m, 4H),
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2.52 (m, 7H), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.86, 157.55, 155.35, 149.54, 142.96, 136.41, 128.18, 127.99, 127.13, 111.94, 54.72, 46.82, 45.52, 43.20, 26.18. HR-MS (ESI): Calcd. C19H22N6O, [M+H]+m/z: 351.1933, found: 351.1932.
4.2.19 8-Benzyl-4-(4-methylpiperazin-1-yl)pteridin-7(8H)-one (21)
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White solid, m. p. 141-142 oC, yield 73%. 1H NMR (400 MHz, Chloroform-d) δ 8.39 (s, 1H), 8.04 (s, 1H), 7.50-7.52 (m, 2H), 7.29 (m,2H), 7.23 (m, 1H), 5.57 (s, 2H), 4.32 (m, 4H), 2.55 (m, 4H), 2.35 (s, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 157.62,
156.52, 155.30, 149.49, 144.21, 136.22, 128.22, 127.63, 127.12, 113.55, 54.69, 46.99,
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337.1777.
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45.48, 43.40. HR-MS (ESI): Calcd. C18H20N6O, [M+H]+m/z: 337.1777, found:
4.3 Antiproliferative activity assays Exponentially growing cells were seeded into 96-well plates at a concentration of 3,000 cells per well. After 24 h of incubation, the culture medium was removed and fresh medium containing various concentrations of the candidate compounds was added to each well. The cells were then incubated for 48 h, thereafter MTT assays were performed and cell viability was assessed at 570 nm by a microplate reader (Biotech, Shanghai, China). 19
ACCEPTED MANUSCRIPT 4.4 Hoechst 33258 staining MKN-45 cells were seeded into a 6-well plate (2×105/well) and incubated overnight for adherent and treated with compound 12 at different concentration for 24 h, and underwent Hoechst 33258 staining for 30 min in the dark. The cells were observed
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under a Nikon Eclipse TE 2000-S fluorescence microscope (Nikon, Japan).
4.5 Cell apoptosis assay
MKN-45 cells were seeded into a 6-well plate (2×105/well) and incubated for 24 h.
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Then the cells were treated with different concentrations of the tested compound 12 for 24 h. Thereafter, the cells were collected and the Annexin-V-FITC/PI apoptosis kit
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(Biovision) was used according to the manufacturer’s protocol. The cells were analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific, MA).
4.6 Western blot analysis
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MKN-45 cells were treated with different concentrations of compound 12 for 24 h, the cells were collected, lysed in RIPA buffer contained a protease inhibitor cocktail for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4 oC. After the
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collection of supernatant, the protein concentration was detected using a bicinchoninic acid assay kit (Beyotmie Biotechnology, Haimen, China). After added with loading buffer, cell lyses were boiled for 10 min at 100 oC for SDS- polyacrylamide gel
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electrophoresis (PAGE). Proteins were transferred to nitrocellulose (NC) membranes. Then the membranes were blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4o C with primary antibodies. After washing the membrane with the secondary antibody (1: 5000) at room temperature for 2 h. Finally, the blots were washed in TBST/TBS. The antibody-reactive were revealed by enhanced chemiluminescence (ECL) and exposed on Kodak radiographic film.
Acknowledgement This work was supported by National Key Research Programs of Proteins (No. 20
ACCEPTED MANUSCRIPT 2016YFA0501800); National Natural Science Foundation of China (Project No. 81430085, 81703326 and 21372206); Key Research Program of Henan Province (No. 1611003110100); the Starting Grant of Zhengzhou University (No. 32210533).
References pteridine derivatives, Res J. Pharm. Biol. Chem. Sci. 6 (2015) 899-904.
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[1] A. Amjed, Ai-Diksin, K. Hanoy, Ai-Amood, Syntheis and biological activity study of some new [2] G. Ferrand, H. Dumas, J.C. Depin, Y. Quentin, Synthesis and potential antiallergic activity of new pteridinones and related compounds, Eur. J. Med. Chem. 31 (1996) 273-280.
[3] Yoshitsugu Kokuryo, Takuji Nakatani, Makoto Kakinuma, Mikio Kabaki, Kyozo Kawata, Akira
M AN U
SC
Kugimiya, Kenji Kawada, Mitsunobu Matsumoto, Ryuji Suzuki, Mitsuaki Ohtani, New γ-fluoromethotrexates modified in the pteridine ring: synthesis and in vitro immunosuppressive activity, Eur. J. Med. Chem. 35 (2000) 529-534. [4] Eleni Pontiki, Dimitra Hadjipavlou-Litina, Alexandros Patsilinakos, Trang M Tran, Charles M Marson, Pteridine-2,4-diamine derivatives as radical scavengers and inhibitors of lipoxygenase that can possess anti-inflammatory properties, Future. Med. Chem. 7 (2015) 1937-1951. [5] A. Guirado, J.I. Lopez Sanchez, A.J. Ruiz-Alcaraz, P. Garcia-Penarrubia, D. Bautista, J. Galvez, First synthesis and biological evaluation of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines as inhibitors of TNF-alpha and IL-6, Eur. J. Med. Chem. 66 (2013) 269-275.
[6] G.A. Breault, J. Comita-Prevoir, C.J. Eyermann, B. Geng, R. Petrichko, P. Doig, E. Gorseth, B. Noonan, Exploring 8-benzyl pteridine-6,7-diones as inhibitors of glutamate racemase (MurI) in gram-positive
TE D
bacteria, Bioorg. Med. Chem. Lett. 18 (2008) 6100-6103. [7] R.C. Reynolds, S. Srivastava, L.J. Ross, W.J. Suling, E.L. White, A new 2-carbamoyl pteridine that inhibits mycobacterial FtsZ, Bioorg. Med. Chem. Lett. 14 (2004) 3161-3164. [8] M.H. Li, S.K. Choi, T.P. Thomas, A. Desai, K.H. Lee, A. Kotlyar, M.M. Banaszak Holl, J.R. Baker, Jr., Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting,
EP
Eur. J. Med. Chem. 47 (2012) 560-572. [9] Z. Zhang, J. Wu, F. Ran, Y. Guo, R. Tian, S. Zhou, X. Wang, Z. Liu, L. Zhang, J. Cui, J. Liu, Novel 8-deaza-5,6,7,8-tetrahydroaminopterin derivatives as dihydrofolate inhibitor: design, synthesis and
AC C
antifolate activity, Eur. J. Med. Chem. 44 (2009) 764-771. [10] S.A. Langie, S. Achterfeldt, J.P. Gorniak, K.J. Halley-Hogg, D. Oxley, F.J. van Schooten, R.W. Godschalk, J.A. McKay, J.C. Mathers, Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring, Faseb J. 27 (2013) 3323-3334. [11] K.K. Liu, S. Bagrodia, S. Bailey, H. Cheng, H. Chen, L. Gao, S. Greasley, J.E. Hoffman, Q. Hu, T.O. Johnson, D. Knighton, Z. Liu, M.A. Marx, M.D. Nambu, S. Ninkovic, B. Pascual, K. Rafidi, C.M. Rodgers, G.L. Smith, S. Sun, H. Wang, A. Yang, J. Yuan, A. Zou, 4-methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors, Bioorg. Med. Chem. Lett. 20 (2010) 6096-6099. [12] W. Zhou, X. Liu, Z. Tu, L. Zhang, X. Ku, F. Bai, Z. Zhao, Y. Xu, K. Ding, H. Li, Discovery of pteridin-7(8H)-one-based irreversible inhibitors targeting the epidermal growth factor receptor (EGFR) kinase T790M/L858R mutant, J. Med. Chem. 56 (2013) 7821-7837. [13] P.A. Roethle, R.M. McFadden, H. Yang, P. Hrvatin, H. Hui, M. Graupe, B. Gallagher, J. Chao, J. Hesselgesser, P. Duatschek, J. Zheng, B. Lu, D.B. Tumas, J. Perry, R.L. Halcomb, Identification and 21
ACCEPTED MANUSCRIPT optimization of pteridinone Toll-like receptor 7 (TLR7) agonists for the oral treatment of viral hepatitis, J. Med. Chem. 56 (2013) 7324-7333. [14] Z.H. Li, X.Q. Liu, P.F. Geng, F.Z. Suo, J.L. Ma, B. Yu, T.Q. Zhao, Z.Q. Zhou, C.X. Huang, Y.C. Zheng, H.M. Liu, Discovery of [1,2,3]Triazolo[4,5-d]pyrimidine Derivatives as Novel LSD1 Inhibitors, ACS Med. Chem. Lett. 8 (2017) 384-389. [15] Z.H. Li, D.X. Yang, P.F. Geng, J. Zhang, H.M. Wei, B. Hu, Q. Guo, X.H. Zhang, W.G. Guo, B. Zhao, B. Yu, L.Y. Ma, H.M. Liu, Design, synthesis and biological evaluation of [1,2,3]triazolo[4,5-d]pyrimidine
RI PT
derivatives possessing a hydrazone moiety as antiproliferative agents, Eur. J. Med. Chem. 124 (2016) 967-980.
[16] Z.H. Li, J. Zhang, X.Q. Liu, P.F. Geng, J.L. Ma, B. Wang, T.Q. Zhao, B. Zhao, H.M. Wei, C. Wang, D.J. Fu, B. Yu, H.M. Liu, Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy, Eur. J. Med. Chem. 135 (2017) 204-212.
[17] S. Wang, L.J. Zhao, Y.C. Zheng, D.D. Shen, E.F. Miao, X.P. Qiao, L.J. Zhao, Y. Liu, R. Huang, B. Yu, H.M.
SC
Liu, Design, synthesis and biological evaluation of [1,2,4]triazolo[1,5-a]pyrimidines as potent lysine specific demethylase 1 (LSD1/KDM1A) inhibitors, Eur. J. Med. Chem. 125 (2017) 940-951. [18] Y.C. Duan, Y.C. Ma, E. Zhang, X.J. Shi, M.M. Wang, X.W. Ye, H.M. Liu, Design and synthesis of novel
M AN U
1,2,3-triazole-dithiocarbamate hybrids as potential anticancer agents, Eur. J. Med. Chem. 62 (2013) 11-19.
[19] R.S. Harapanhalli, R.W. Howell, D.V. Rao, Bis-benzimidazole dyes, Hoechst 33258 and Hoechst 33342: radioiodination, facile purification and subcellular distribution, Nucl. Med. Biol. 21 (1994) 641-647.
[20] Z. Chen, X. Liang, H.Y. Zhang, H. Xie, J.W. Liu, Y.F. Xu, W.P. Zhu, Y. Wang, X. Wang, S.Y. Tan, D. Kuang, X.H. Qian, A New Class of Naphthalimide-Based Antitumor Agents That Inhibit Topoisomerase II and
TE D
Induce Lysosomal Membrane Permeabilization and Apoptosis, J. Med. Chem. 53 (2010) 2589-2600. [21] X.J. Shi, B. Yu, J.W. Wang, P.P. Qi, K. Tang, X. Huang, H.M. Liu, Structurally novel steroidal spirooxindole by241 potently inhibits tumor growth mainly through ROS-mediated mechanisms, Sci.
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Rep. 6 (2016) 31607.
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ACCEPTED MANUSCRIPT
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Highlights The pteridin-7(8H)-one derivatives showed potent inhibition against the cancer cells. Compound 12 exerted the most potent and broad-spectrum antiproliferative activity. Compound 12 induced the apoptosis and G2/M arrest of MKN-45 cells. A novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold was constructed.